REVIEW ARTICLE

published: 27 March 2014

doi: 10.3389/fphar.2014.00051

Mechanisms of cellular fibrosis associated with cancer

regimen-related toxicities
Maria L. Mancini* and Stephen T. Sonis
Biomodels LLC, Watertown, MA, USA

Edited by: Fibrosis is a common, persistent and potentially debilitating complication of chemotherapy
Lynne Anne Murray, MedImmune Ltd, and radiation regimens used for the treatment of cancer. The molecular mechanisms
UK
underlying fibrosis have been well studied and reveal overall processes that are largely
Reviewed by:
Darryl Knight, University of
ubiquitous. However, it is important to note that although the processes are similar,
Newcastle, Australia they result in cellular phenotypes that are highly tissue specific. These tissue specific
Lynne Anne Murray, MedImmune Ltd, differences may present opportunities for therapeutic interventions to prevent or treat this
UK often irreversible condition. Data generated from animal models of cancer therapy-related
*Correspondence: tissue toxicities have revealed that the signaling pathways involved in fibrosis are the same
Maria L. Mancini, Biomodels LLC, 313
Pleasant Street, Watertown,
as those involved in the normal injury response and include the transforming growth
MA 02472, USA factor β superfamily and a range of pro-inflammatory cytokines. The critical difference
e-mail: mmancini@biomodels.com between normal wound healing and fibrosis development appears to be, that in fibrosis,
these signaling pathways escape normal cellular regulation. As a result, an injury state
is maintained and processes involved in normal healing are usurped. There are a few,
if any, therapeutics that effectively prevent or treat fibrosis in patients. Consequently,
cancer survivors may be chronically plagued with a variety of life-altering fibrosis-related
symptoms. Uncovering the signaling pathways that drive cellular fibrosis is paramount
to the development of specific therapeutics that will mitigate this potentially devastating
condition.
Keywords: fibrosis, radiation therapy, chemotherapy toxicity, oral mucositis, radiation dermatitis, proctitis,
pulmonary fibrosis, TGF-ß

INTRODUCTION with cellular fibrosis of epithelial tissues and include radiation

Radiation and chemotherapy remain the most commonly used
therapies for the treatment of multiple types of human cancer.
While these therapies have been met with great success in the treat-
ment of tumors, they are known to induce a wide range of acute
and chronic toxicities. These regimen-related toxicities are not
only associated with poor health outcomes, but they often become
dose-limiting for patients and impair patients’ quality of life (QoL)
and recovery in both the short and the long term (Elting et al.,
2008). Hematological disorders such as anemia, thrombocytope-
nia, and neutropenia are among the most common complications
associated with radiation and chemotherapy; however, cancer
patients are also at risk for a wide range of non-hematological tox-
icities (Sonis and Keefe, 2013). The overall incidence of some form
of cancer treatment-related toxicity is almost 100% and can occur
both during cancer treatment (acute toxicities), or will develop
well after the completion of treatment (≥100 days, late toxicities).
Understanding chemotherapy and radiation induced toxicities is
of high importance due to their direct impact on patients’ symp-
toms and QoL and their high resource and financial burden. This
review will focus on regimen-related toxicities that are associated
Abbreviations: TGFβ, transforming growth factor β; QoL, quality of life; RT, radi-
ation therapy; ROS, reactive oxygen species; SOD, superoxide dismutase; CTGF,
connective tissue growth factor; TNF-α, tumor necrosis factor alpha; INF-γ, inter-
feron gamma; EMT, epithelial to mesenchymal transition; Gy Gray, a unit of
absorbed dose of ionizing radiation.
and/or chemotherapy-induced fibrosis of the gastrointestinal tract
(oral mucositis and proctitis), the skin (dermatitis), and the lung
(pulmonary fibrosis).
MECHANISMS OF FIBROSIS
Within the past decade, there has been a major shift in the
conventional paradigms associated with the pathogenesis of
regimen-related toxicities in cancer patients. Historically, normal
tissue damage was attributed to the concept that since radiation
or chemotherapy could not distinguish between rapidly divid-
ing cancer cells and rapidly dividing normal cells the result was
non-specific clonogenic cell death. Not only were the cellular
kinetics associated with normal tissue toxicity inconsistent with
this hypothesis, but it failed to address damaging changes to
peripheral tissue such as subepithelial connective tissue or muscle
(i.e., heart) and completely ignored non-tissue based complica-
tions like fatigue, cognitive dysfunction or cachexia. Accumulating
evidence suggests that, while some clonogenic cell death does
occur, the bulk of pathogenesis is the consequence of a sequence of
related biological events that result in both direct and indirect tis-
sue and systemic damage mediated by a diverse range of canonical
pathways. Of particular interest has been the finding that the tem-
poral genomic characterization of these toxicities has shown their
compatibility with conditions having similar phenotypes includ-
ing chemotherapy-induced diarrhea and inflammatory bowel

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Mancini and Sonis
disease (IBD), chemotherapy-fatigue and chronic fatigue syn-
drome, regimen-related cognitive dysfunction, and Alzheimer’s
disease. While this observation has not been surprising, it does
serve to emphasize the potential impact of better understanding
fibrosis in the context of cancer treatment, as well as possibly open-
ing treatment intervention opportunities beyond the oncology
population.
Data outlining molecular mechanisms by which treatment-
related fibrosis develops has largely been captured from animal
models that accurately replicate chemotherapy or radiotherapy
regimens routinely used in patients (Sonis and Keefe, 2013).
Immediately following insult due to chemotherapy and radia-
tion, there is a large cellular response that involves cell type
specific programs occurring in three general phases (Figure 1).
First is the inflammatory phase, where inflammatory cells are
recruited and release cytokines to recruit fibroblasts and other
immune cells to the site of injury. Second is the proliferative phase,
which is characterized by fibroblasts proliferating and migrating
to the site of injury where they form a scaffold on a temporary
fibronectin matrix present in the tissue and deposit collagen type
III to form a new barrier. Third is the remodeling phase which
lasts several weeks and involves building up the new extracellular
FIGURE 1 | Mechanisms of radiation and chemotherapy-induced fibrosis. Above is an illustration of the processes that drive fibrosis detailing the
mechanisms governing the overall tissue changes that occur as a result of radiation and chemotherapy induced injury; progressing from normal tissue to an
eventual fibrotic state.
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Cancer regimen-induced fibrosis
matrix (ECM) by the converting the flexible collagen type III into
the more permanent collagen type I. This conversion is mediated
through both secreted proteases and matrix building proteins from
local fibroblasts. Not surprisingly, heterogeneity exists among the
various fibroblast populations recruited; fibroblast subsets have
specialized functions and vary in rates of proliferation, response
to inflammatory signals and ECM production (Sempowski et al.,
1995).
Injury resulting from radiation and chemotherapy is initiated
through two major paths: radiolytic hydrolysis and stimulation
of the innate immune response. Of the two, oxidative stress is
the best studied with respect to cancer treatment-associated tis-
sue injury. Radiation or chemotherapy-induced oxidative stress
leads to the production of oxygen free-radicals; specifically the
reactive oxygen species (ROS) superoxides, hydrogen peroxides,
and hydroxyl radicals that cause oxidative damage to the tissue
(Pan et al., 2012). Once damage to the tissue has been initiated,
inflammatory cells are recruited to the injured area, a process
orchestrated by vasodilation and vascular permeability. On the
cellular level, fibrosis involves the coordination of a variety of
cell types largely mediated through the fibroblast. The infiltrat-
ing immune cells secrete cytokines that drive the differentiation of
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fibroblasts and other self-renewing cells into myofibroblasts which
deposit collagens and other ECM proteins at and around the site
of tissue damage (Eckes et al., 2000; Wang et al., 2011). Under nor-
mal circumstances of wound repair, the expanded ECM would be
provisional until the process of re-epithelialization occurs. When
the wound repair process is deregulated, and re-epithelialization
is prevented, fibrosis occurs (Eckes et al., 2000; Ueha et al., 2012).
Until recently it was generally accepted that fibrosis occurs in a con-
fined space only affecting the area immediately surrounding the
site of injury. It has since become clear that fibrosis arises and per-
sists systemically. Circulating immune cells, chemokines, and bone
marrow derived fibroblasts are recruited to sites of injury gen-
erating and depositing excess ECM proteins (Andersson-Sjöland
et al., 2011). On a molecular level, the processes of fibrosis are
driven by transforming growth factor beta (TGFβ), connective tis-
sue growth factor (CTGF), tumor necrosis factor alpha (TNF-α),
and interferon gamma INF-γ (Reviewed in Leask and Abraham,
2004). Susceptible organs include any connective tissue contain-
ing organ and/or tissues containing mesenchymal cells that are
capable of differentiating into fibroblasts. While the initial insult
is often focused on a specific organ/tissue, systemic effects are seen
in distant sites highlighting the widespread nature of regimen-
related toxicities. For example patients receiving radiation therapy
(RT) with or without cytotoxics for head and neck cancer develop
diarrhea, supporting the idea that even directed therapies are
not contained to the treated tissues (Vermorken and Specenier,
2010).
The TGFβ signaling axis drives the majority of the cellular
events associated with radiation-induced fibrosis (Boerma et al.,
2013). The TGFβ superfamily regulates a wide variety of cellular
processes in response to injury including survival, proliferation,
and migration (Reviewed in Massague, 2012). TGFβ binds to its
cognate receptors and channels its instructive signals through the
Smad family of transcription factors inducing the expression of
target genes involved in the cellular phenotypes described above
and also upregulates the genes responsible for collagen synthesis
(Ghosh et al., 2008). In addition, signaling through this path-
way drives cellular dedifferentiation and reprogramming. Multiple
studies demonstrate that epithelial cells can undergo epithelial
to mesenchymal transition (EMT), acquire the characteristics
of fibroblasts and subsequently differentiate into myofibroblasts.
This transition is driven by transcriptional changes that are medi-
ated by TGFβ signaling and ultimately further exacerbate tissue
fibrosis (Venkov et al., 2007; Wang et al., 2011). Taken together,
TGFβ acts both as a potent chemoattractant for fibroblasts and
subsequently a mediator of proliferation, migration, differentia-
tion, and ECM deposition of the recruited fibroblasts at the site of
injury.
The molecular mechanisms of TGFβ signaling associated with
the development of fibrosis have been underscored through genet-
ically modified animal models (Chen et al., 2001; Flanders et al.,
2002; Chan et al., 2012; Balli et al., 2013). Epithelium in animals
engineered to suppress Smad3 has a reduced fibrotic response
(Flanders et al., 2002; Oh et al., 2012). Furthermore, Smad3
null animals demonstrate accelerated wound repair characterized
by enhanced re-epithelialization with a reduced inflammatory
response (Ashcroft et al., 1999). While the data from these studies
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Cancer regimen-induced fibrosis
suggest that TGFβ/Smad signaling is directly involved in the induc-
tion of fibrosis, other studies have shown that signaling through
this pathway is not required for maintaining fibrosis and therefore
therapies directed at regulating Smad activity would likely have
to be administered as an early intervention (Holmes et al., 2001;
Leask et al., 2003). To further support this notion, the findings
of a recent meta-analysis of clinical data failed to demonstrate an
association between three SNPs commonly associated with TGFβ1
and the risk of late radiation-induced normal tissue damage (Zhu
et al., 2013).
CTGF is induced by TGFβ, specifically via Smad enhancer ele-
ments in the CTGF promoter, and therefore acts as a downstream
effector of TGFβ mediated fibrosis (Leask et al., 2003). Data suggest
that after initial induction, expression of CTGF remains constitu-
tive in areas of fibrosis and escapes regulation by TGFβ signaling
(Holmes et al., 2001). Therefore cellular events downstream of
CTGF secretion, including induction of collagen I expression in
neighboring cells, may contribute to the maintenance of fibrosis.
Furthermore, the paracrine nature of CTGF activity suggests a
pivotal role in inducing fibrosis in surrounding tissues further
exacerbating the duration and severity of fibrotic phenotypes
(Sonnylal et al., 2013). One mechanism of negative regulation
of these signaling events involves the pro-inflammatory cytokine
TNF-α which acts as an inhibitor of TGFβ induced CTGF expres-
sion and therefore has anti-fibrotic activity (Mori et al., 2002).
In addition, endogenous TNF-α applied directly to injured skin
reduced the deposition of collagen. Some data suggests however,
in certain situations sustained TNF-α expression may contribute
to fibrosis in alternative mechanisms, through directly stimulat-
ing the proliferation of fibroblasts (Piguet et al., 1990; Leask and
Abraham, 2004). That TNF-α does not regulate CTGF expression
directly further supports the importance of CTGF related signaling
events downstream of TGFβ in propagating fibrosis.
Increased expression of the pro-inflammatory cytokine INF-γ
goes hand in hand with tissue fibrosis. Naïve CD4+ T helper (Th)
cells differentiate into Th1 and Th2 subsets that secrete cytokines
in response to inflammation including INF-γ and a plethora of
interleukins essential for immune cell functions (Chen et al., 2001).
The specific role of INF-γ is incompletely understood; it is unclear
whether the presence of INF-γ at sites of fibrosis confers a role
that is promoting or reparative. One study demonstrated that
animals deficient in INF-γ did not develop pulmonary fibrosis
resulting from treatment with Bleomycin suggesting a critical role
for this cytokine in promoting fibrosis (Chen et al., 2001). By con-
trast, other studies have shown that INF-γ has anti-fibrotic activity
mediated through suppression of collagen synthesis by fibroblasts
and inhibition of TGFβ expression (Gurujeyalakshmi and Giri,
1995). Therefore, data suggest that spatiotemporal expression of
INF-γ may determine the role it plays in the development and
maintenance of fibrosis. This is similar to the dichotomous role
for TNF-α, the pro-inflammatory cytokine that is also regulated by
and synergizes with INF-γ (Nathan et al., 1984). Taken together the
signaling events regulating fibrosis represent distinct yet interde-
pendent signaling pathways that become de-regulated in situations
of radiation and chemotherapy-induced injury. These pathways
provide options for therapeutic interventions (Table 1) and will
be categorically explained in greater detail.
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Table 1 | Current standard of care therapies for common regimen-related toxicities.
Regimen-related toxicity Treatment
Oral mucositis MuGuard, gelclair
Caphosol, kepivance
Proctitis 5-ASA, steroids
Sucralfate, metronidazole
Dermatitis Aloe vera
Pulmonary fibrosis Amifostine
Superoxide dismutases (SODs)
Examples of therapies used to treat patients suffering with the toxicities described in this article. None of these therapies are specifically targeted to address fibrosis
directly; rather they are designed to prevent the initial inflammatory responses that eventually lead to fibrosis for purposes of prevention. This strategy has been met
with limited success and therefore there is an unmet clinical need for development of therapeutics focused specifically on fibrosis.
FIBROSIS ASSOCIATED WITH ORAL MUCOSITIS
Radiotherapy is a treatment mainstay for cancers of the head and
neck and is typically administered concomitantly with radiosen-
sitizing doses of chemotherapy, most commonly Cisplatin (Sonis,
2011). The oral, oropharyngeal, hypopharyngeal, and laryngeal
mucosa are often included in the radiation field. As a consequence
severe tissue injury in the form of mucositis is virtually ubiquitous.
Lesions of oral mucositis consist of diffuse, deep, extremely painful
ulcers involving the mouth’s movable mucosa (Sonis, 2013). As in
most treatment paradigms, radiation is in small, daily fractionated
doses of 2Gy, 5 days per week, for cumulative doses of 60–70Gy
(Silverman, 2007). Chemotherapy is given either every 3 weeks
during the radiation period (days 0, 21, and 42) or weekly, but
in smaller doses. These treatment schemes induce a predictable
pattern of mucositis. By the end of the first week of treatment
(cumulative radiation dose of 10Gy) erythematous changes start
to occur and are accompanied by a level of pain described as being
comparable to a bad food burn. By cumulative doses of 30Gy,
ulceration develops (Silverman, 2007; Sonis, 2013). Unlike the
typical mouth sores of aphthous stomatitis (canker sores), ulcer-
ative lesions associated with mucositis are more broad and deep.
Consequently, they are disproportionately painful requiring opi-
oid analgesics which are often ineffective. Patients frequently are
unable to eat by mouth and require gastrostomy tube placement
for feeding (Sonis, 2007). Subepithelial changes that accompany
mucositis predispose patients to fibrosis and the clinical devel-
opment of trismus, which is characterized by a restricted ability
to open the mouth sometimes referred to as “lock jaw” (Lyons
et al., 2013). Of regimen-related tissue injuries, the pathogenesis
of mucositis is probably best understood and has been described
as a 5-phase algorithm which it appears to share with other types
of cancer regimen-induced epithelial damage (Sonis et al., 2000).
While originally directed at the oral mucosa, it is now clear that
the biological sequence is the same for regimen-related damage
throughout the gastrointestinal tract.
As noted above, the initiation phase is characterized by oxida-
tive stress and activation of the innate immune response which
Frontiers in Pharmacology | Inflammation Pharmacology
Cancer regimen-induced fibrosis
Mechanism
Promotes barrier function
Topical biologics designed to promote healing
Anti-inflammatory
Promotes barrier function, prevents bacterial colonization
Largely unknown; limited barrier function and lubrication
Produces cytoprotective thiols
Dismutate superoxides; prevents oxidative damage
largely occurs in the endothelium and connective tissue of the
submucosa after exposure to radiation (Sonis, 2007). In response
to the activation of a range of canonical pathways, a cytokine cas-
cade follows as does simultaneous fibronectin breakdown, and
amplification of pro-inflammatory cytokine signaling cascades in
the aforementioned TGFβ and TNF-α pathways. Ultimately the
epithelium breakdowns down and ulceration occurs (Sonis et al.,
2000). Secondary bacterial colonization and breaks in the mucosal
barrier allow for penetration of whole bacteria or, more com-
monly, cell wall products which activate infiltrating macrophages
to produce additional cytokines. The injured mucosa undergoes
extensive remodeling to seal off the tissue to try and prevent inva-
sion of the bacteria. This process involves cellular deposits of ECM
that, as with many situations of wound repair, can encompass the
surrounding tissue and result in extensive fibrosis.
Clinically, patients with fibrosis of the mouth develop trismus
and cannot function normally (Lyons et al., 2013). But fibrosis
is not limited to the mouth. Salivary function is obscured by
replacement of parenchyma with fibrous tissue and esophageal
strictures can develop (Fujita-Yoshigaki and Qi, 2009). While
mucositis is an acute toxicity, fibrosis-related changes tend to
be more chronic and are thus increasingly significant as cancer
survivorship improves. Animal models of mucositis in rats, mice
and hamsters have provided highly useful templates to define the
pathogenesis of mucosal injury and evaluate the potential effi-
cacy of new interventions. Among the models routinely used, a
highly translatable model in hamsters has been especially useful in
drug development and in demonstrating the relationship between
radiation-induced mucosal damage and the development of fibro-
sis (Ara et al., 2008; Murray et al., 2010). An approved, effective
mechanistically based therapy for radiation-induced mucositis is
being aggressively sought. It is likely that a halo benefit from such
a treatment will be attenuation of fibrosis development.
FIBROSIS ASSOCIATED WITH PROCTITIS
Radiation-induced proctitis is a complication resulting from
radiation directed at the lower abdomen or pelvis typically
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Mancini and Sonis
associated with rectal, prostate, or cervical malignancies. The
incidence of proctitis is approximately 75% with symptoms
consisting of rectal bleeding, mucus production and diarrhea
(Counter et al., 1999). Not surprisingly given the nature of rec-
tal epithelium, symptoms begin within 2–3 weeks after the start
of treatment. In contrast to lesions of the upper gastrointestinal
tract, however, proctitis often lasts anywhere from a few months
to several years following the completion of radiation therapy.
The stages are multifactorial beginning with initial damage to
the mucosa, followed by delayed slow growing connective tis-
sue and finally a tissue response to vascular ischemia (Okunieff
et al., 2005). In addition to rectal bleeding and mucus, patients
suffering from proctitis also experience tenesmus, or a feeling
or inability to empty the bowel upon defecation. This occurs
as a result of epithelial fibrosis in the rectum due to radiation
exposure, a condition which is often permanent and irreversible
(Symon et al., 2010).
The overall pathogenesis of fibrosis associated with radiation-
induced proctitis is similar to that which occurs in oral mucositis.
However, unlike oral mucositis, radiation-induced proctitis has
not been as aggressively studied pre-clinically and there exists a
very high unmet clinical need in this area. Proctitis is initiated
on the cellular level with apoptosis, disruptions of mitosis, and
fibroblastic proliferation that leads to swelling and sloughing of
the rectal mucosa (Haboubi et al., 1988; Brunn and Fletcher, 2006).
It has become increasingly clear that the complications associ-
ated with proctitis involve coordination between the processes
of fibrosis and angiogenesis. The cellular alterations to the vas-
culature including neovascularization and telangiectasias lead to
clinical symptoms of persistent bleeding. Increased fibrosis causes
ischemia and eventual necrosis of the bowel tissue (Haselton et al.,
1985; Fajardo, 2005). The exact mechanisms for the late changes
in vascularity and fibrosis have yet to be elucidated, however, there
is evidence that several growth factors including platelet-derived
growth factor, vascular endothelial growth factor, and fibroblast
growth factor play key roles in the pathology (Brunn and Fletcher,
2006) More recent evidence has suggested a role for mast cell
involvement based on the synergistic expression of endothelial and
inflammatory genes in response to radiation, including p38a MAP
kinase and p65 (NF-κB; Blirando et al., 2011). Given the findings
that serine peptidases, particularly tryptases, secreted by mast
cells are able to stimulate both fibroblast chemotaxis and collagen
production, the potential significance of mast cells in the patho-
genesis of fibrosis definitely warrants additional investigation
(Hugh and Pemberton, 2002; Caughey, 2007). Furthermore, radi-
ation induced an increase in expression of αv β3 integrin (Abdollani
et al., 2005). The αv β3 is highly expressed on endothelial cells and
is known to have potent angiogenic activity involving prolifera-
tion and migration of endothelial cells mediated through FGF and
therefore likely contributes to the vascular changes observed in
radiation proctitis (Son et al., 2013). Taken together, these mech-
anisms may individually or collectively contribute to long term
defects in tissue and vascular integrity associated with propagat-
ing the injury response mechanisms that promote fibrosis (Sheth
et al., 2009).
Current treatment options for radiation proctitis vary greatly
in success rates (Leiper and Morris, 2007). While there are few, if
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Cancer regimen-induced fibrosis
any, compelling clinical studies in the treatment of radiation proc-
titis, the majority of therapies are based on the results of small
unblinded studies with mixed results. Animal models that utilize
focused radiation to the bowel have been effective tools for screen-
ing potential compounds because the inflammatory responses that
occur in these models accurately represent the diversity seen in
patients (Skwarchuk and Travis, 1998; Kang et al., 2000; Symon
et al., 2010). The most common first-line therapies have been
adapted from the treatments used in IBD, including the anti-
inflammatory compounds 5-ASA and steroids, sucralfate which
promotes barrier function and metronidazole to mitigate bacterial
colony formation (Symon et al., 2010). Increasingly, endoscopic
therapies are being employed to control bleeding which include
heat probes, lasers, and most commonly argon plasma coagu-
lation (APC). APC involves the flow ionized argon gas and can
target small or larger areas of bleeding without making physical
contact to the tissue (Leiper and Morris, 2007). Other studies
suggest that long term suppression of high pro-inflammatory
cytokine levels while also stimulating with pro-survival growth
factors will provide the right balance to prevent long term com-
plications (Okunieff et al., 2005). Linard et al. (2013) recently
reported a different therapeutic approach focusing on prevent-
ing the progression of fibrosis with autologous mesenchymal stem
cells (MSCs). Using an irradiated pig model, they initiated infu-
sion of MSCs following endoscopic identification of early fibrosis.
MSCs limited the progression of radiation-induced fibrosis due to
a reduction in collagen deposition, a decreased transforming TGFβ
response and modification of matrix metalloproteinase/TIMP bal-
ance. The novelty of this tactic provided a new arena for developing
potential therapeutic strategies that may address these currently
irreversible complications.
FIBROSIS ASSOCIATED WITH RADIATION DERMATITIS
Fibrosis of the skin in the context of cancer treatment occurs
as a result of radiation-induced dermatitis. Radiation dermatitis
is a common side effect of radiation therapy. It has a reported
frequency of 85% and is commonly seen in patients receiving
radiotherapy for the treatment of the breast, lung, prostate or
head and neck cancers. The fibrosis which often accompanies
dermatological changes, results in symptoms similar to contrac-
tures that markedly compromise patients’ ability to move freely.
Examples include patients treated for breast cancer have a lim-
ited range of motion of their arm, or head and neck cancer
patients who have hindered head movement. The clinical presenta-
tion varies from mild erythema to complete epithelial breakdown
manifested by painful ulceration and fibrosis (Ryan, 2012). Injury
to the skin occurs immediately following the first dose of radi-
ation, disrupting the self-renewing cells of the epidermis (Ryan,
2012). Subsequent doses prevent this cell population from fully
replenishing itself and also elicit an inflammatory response result-
ing in systemic complications. Later doses further exacerbate the
alterations in cellular phenotypes and can cause chronic issues
including delayed ulcerations, fibrosis and in the most severe cases,
necrosis of the tissue (Bey et al., 2010; Salvo et al., 2010).
After an initial exposure to radiation, there is immediate dam-
age to the keratinocyte cells of the skin, which is accompanied
by a simultaneous increase in free-radicals, DNA damage and
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inflammation (Brown and Rzucidlo, 2011; Ryan, 2012). Like other
epithelial tissues, many of these biological changes are apparent in
the dermis. The TGFβ signaling pathway is largely implicated in
driving the pathogenesis of radiation-induced dermatitis where
TGFβ levels are markedly increased in irradiated skin and remain
elevated for a long period of time (Martin et al., 2000; Flanders
et al., 2003). TGFβ isoforms act as potent chemotactic factors for
monocytes, neutrophils, mast cells, and fibroblasts. Furthermore,
the Smad family of proteins regulates expression of additional
genes responsible for inflammation and fibrosis amplifying and
maintaining the cascade of inflammatory signals (Flanders et al.,
2002). Smad3 null mice exposed to ionizing radiation exhibited
decreased skin damage and reduced fibrosis compared to wild
type animals, further supporting a critical role for this signaling
pathway in the phenotypes associated with radiation dermatitis
(Flanders et al., 2002, 2003).
There is currently no effective intervention to prevent or favor-
ably modify the course and severity of radiation dermatitis (Wong
et al., 2013). Treatment strategies have been based on those associ-
ated with thermal burns. Consequently, there is an unmet clinical
need to develop specific therapies to target the mechanisms under-
lying the development and persistence of radiation dermatitis
(Lataillade et al., 2007; Bey et al., 2010; Salvo et al., 2010; Chan
et al., 2012). Pre-clinical animal models of radiation-induced der-
matitis in the published literature are limited however, a few
recent studies performed in mouse models have shown promis-
ing therapeutic efficacy signals. In one example, treatment with a
Toll-like receptor 5 agonist reduced radiation dermatitis associated
epidermal hyperplasia and dermal inflammation through activa-
tion of endogenous antioxidants reducing free-radicals (Burdelya
et al., 2011). In another study, the antiallergic agent Azelastine
was administered in the food of mice that received an acute dose
of radiation and reduced the severity of radiation dermatitis. In
these animals there was a modest reduction in the severity of der-
matitis. The authors proposed that the mechanism responsible
was increased epithelial cell stabilization post-radiation exposure
due to preventing the influx of Ca2+ into the cell resulting in a
decrease in free radical generation (Murakami et al., 1997). Until
an effective therapeutic is approved for this indication, patients
will continue to be treated with standard burn agents such as
Aloe Vera which neither prevents fibrosis nor addresses the pal-
liative component associated with skin injury due to radiation
exposure.
The clinical observation of heterogeneity in the manifesta-
tion of fibrosis-related to radiation-induced dermatitis suggests
a possible genetic basis for risk. The results of a recent study
by Andreassen et al. (2013) in which cultured fibroblasts from
head and neck cancer patients with subcutaneous fibrosis were
irradiated. Analysis of the cells provided validation of a group of
overexpressed genes associated with a positive fibrosis phenotype.
Additional studies on the predictive ability of synergistically acting
genes, both in tissue and in peripheral blood, would certainly be
of great interest.
PULMONARY FIBROSIS
Radiation therapy is a critical component in the treatment of many
forms of cancers of the lung, breast, and lymphomas. In these cases,
Frontiers in Pharmacology | Inflammation Pharmacology
Cancer regimen-induced fibrosis
the radiated fields include the thorax which exposes the lungs to
the potential for injury. Radiation directed to the thoracic region
exceeding 50Gy damages pulmonary tissues and may lead to the
development of dose-limiting pneumonitis or fibrosis (Reviewed
in Tsoutsou and Koukourakis, 2006). Moderate to severe radiation
pneumonitis occurs with a reported incidence of between 10 and
20% (Mehta, 2005) and is associated with alveolar damage that
causes a significant inflammatory response in the interstitial space
of the lung. The increased vascular permeability causes edema
and accumulation of proteins in the alveolar space (Rosai, 1996).
Pneumonitis develops within 12 weeks of receiving radiation ther-
apy and symptomatically manifests by shortness of breath. Severe
cases increase the risk of treatment-related death significantly.
Ultimately, pneumonitis may transition to pulmonary fibrosis.
Pulmonary fibrosis is among the most thoroughly studied
cancer regimen-induced forms of fibrosis. Although it can be
caused by a variety of chemotherapeutic drugs or by radiation
and is associated with poor clinical outcomes, its pathogenesis
is incompletely understood. Of chemotherapies associated with
pulmonary fibrosis, cases resulting from bleomycin treatment are
the best studied. Radiation-induced fibrosis has been investigated
in parallel and emerging data suggests the biological pathways
which impact end organ damage are likely to be similar. Radiation-
induced pulmonary fibrosis is mediated through inflammatory
cells that are recruited and accumulate in the interstitial space.
It is believed that these cells are responsible for the majority of
TGFβ production which drives the differentiation of fibroblasts to
myofibroblasts depositing collagens into the ECM (Eckes et al.,
2000; Wang et al., 2011). In addition, the pulmonary alveolar
type II epithelial cells can undergo EMT and differentiate directly
into matrix depositing myofibroblasts (Radisky, 2005; Andersson-
Sjöland et al., 2011). The process of EMT is mediated by Snail and
Twist transcription factors which repress E-Cadherin and promote
a mesenchymal phenotype enabling the epithelial cells to escape
attachment from the basement membrane permitting anchor-
age independent growth and migration into the interstitial space
(Willis et al., 2005); ultimately, the damaged alveoli collapse and
are fully encompassed by connective tissue (Almeida et al., 2013).
The process of fibrosis occurs several months later than pneu-
monitis, typically appearing 6 months after radiation exposure.
The timing of these late effects are supported by data show-
ing that increased TGFβ signaling is sustained after pneumonitis
resolves thus promoting the latent fibrosis (Rube et al., 2000). Sim-
ilar signaling events have been described for bleomycin-induced
pulmonary fibrosis. The common clinical practice of administer-
ing concomitant chemoradiation (in this case, chemotherapy is
administered as a radiosensitizer) increases the risk of severe and
extended pulmonary fibrosis. Mouse models of thoracic radiation
and treatment with bleomycin have revealed molecular mecha-
nisms driving the general induction process of fibrosis in the lung.
In addition to the previously described classical TGFβ and TNF-α
signaling pathways that are responsible for initiating inflammation
at the site of injury, there is subsequent involvement of chemokines
and their receptors in orchestrating the recruitment and trafficking
of the immune cells from the circulation to the sites of inflam-
mation driving the epithelial cell responses within the alveoli
(Johnston et al., 2002; Han et al., 2011). Interestingly, chemokine
March 2014 | Volume 5 | Article 51 | 6
Mancini and Sonis
production differs significantly between the pneumonitic and
fibrotic phases of injury suggesting differential roles for subsets of
chemokine family members and likely specificity in the immune
cells they recruit (Johnston et al., 1998). Whereas chronic inflam-
mation is a hallmark of fibrosis, sustained chemokine recruitment
and trafficking of immune cells may lie at the crux of the cellular
events propagating the long term complications associated with
the condition.
Like the majority of regimen-related toxicities, the risk of pul-
monary fibrosis is not ubiquitous among patients undergoing
treatment. In reality, despite demographic and treatment inten-
sity equivalence, only a relatively small fraction of patients go on
to develop fibrosis. This clinical observation has led to signifi-
cant interest to determine those factors that are associated with
risk. It now appears that genetic differences among patients, espe-
cially those genes that are associated with the pathways leading
to fibrosis are fundamental determinants of risk. This hypothesis
was elegantly demonstrated by Paun and Haston (2012) who com-
pared radiation-induced pulmonary fibrosis susceptibility across
a number of mouse syngeneic mouse strains and then, using a
genome-wide association study format, identified specific SNP loci
associated with the condition. The complexity of the pathobiology
of radiation-induced fibrosis and its impact on clinical risk predic-
tion has been further substantiated by candidate SNP approaches.
As noted above, TGFβ1 has been associated with the biological
sequence leading to fibrosis. Consequently, TGFβ1 SNPs seemingly
made a good target for risk determination. Results of substantive
clinical studies have been, however, inconsistent (Barnett et al.,
2012). It seems likely that effective genomic risk prediction of
fibrosis will rely on the discovery of a cluster of SNPs or genes
(Petrovski et al., 2009).
Preventive strategies such as the use of intensity-modulated
radiation therapy (IMRT) have been suggested to reduce the
frequency and severity of radiation induced pulmonary fibro-
sis (Lavrenkov et al., 2007). On the other hand, modifying
treatment-related strategies associated with chemotherapy is more
challenging given the wide range of drugs that are associated with
lung injury (Carver et al., 2007). Despite attempts to mitigate
cancer therapy induced lung injury, pulmonary fibrosis persists
and remains a significant side effect that is refractory to most
treatments. Presently a lung transplant is viewed as the treat-
ment of choice (Thabut et al., 2003) however, potential biologically
directed targets for intervention are emerging. For example, the
Foxm1 transcription factor has been implicated in driving inflam-
mation, EMT and proliferation of fibroblasts in the lung and
therefore might be of interest as a potential target for intervention
(Balli et al., 2013). Additional treatment strategies for pulmonary
fibrosis are similar to the previously described therapies for fibrosis
from other tissue origins including agents which act as free rad-
ical scavengers. One such example approved by the FDA for use
as a broad spectrum radioprotective agent is the thiol amifostine
(Kouvaris et al., 2007). Clinical trial data suggests that amifostine
protects against the long term effects of radiation and chemora-
diation induced toxicity to lung and soft tissue including fibrosis.
Treatment with amifostine also allows for higher doses of radiation
to be administered in situations where that is required for higher
tumor kill (Koukourakis et al., 2013). Superoxide dismutases
www.frontiersin.org
Cancer regimen-induced fibrosis
(SODs) are naturally occurring enzymes in the body which act
in the early stages of injury. SODs dismutate superoxides to
form hydrogen peroxide (H2 O2 )and catalase further decomposes
H2 O2 to oxygen and water. Because radiation therapy reduces the
endogenous SOD, SOD mimetics have been developed for ther-
apeutic use and have demonstrated success in reducing fibrosis
by enhancing the activity of antioxidant enzymes in the tissue,
preventing oxidative damage with fewer side effects and higher
potency than amifostine (Gao et al., 2012; Pan et al., 2012).
CONCLUDING STATEMENTS
Chronic problems associated with fibrosis are of high impor-
tance because they can ultimately force physicians to limit an
individuals’ cancer treatment due to a decreased tolerance of the
therapies employed. As a result, there is a high demand to develop
interventions designed to prevent or treat fibrosis associated with
regimen-related toxicities. Animal models designed to mimic radi-
ation and chemotherapy-induced fibrosis in patients and to help
understand the exact mechanisms of these pathways have revealed
that there are marked differences in the cytokine responses among
animal strains. This is consistent with a varied cytokine response
seen in patients; therefore highly valuable information has been
gained from collecting and incorporating data from several differ-
ent models. The discovery that many toxicities seem to cluster
suggests shared pathoetiologies that will not only assist in the
development of such therapeutics, but will also likely result in
the use of one therapy for the treatment or prevention of fibrosis
in multiple tissue types.
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 04 November 2013; paper pending published: 13 February 2014; accepted:
09 March 2014; published online: 27 March 2014.
Citation: Mancini ML and Sonis ST (2014) Mechanisms of cellular fibrosis associ-
ated with cancer regimen-related toxicities. Front. Pharmacol. 5:51. doi: 10.3389/
fphar.2014.00051
This article was submitted to Inflammation Pharmacology, a section of the journal
Frontiers in Pharmacology.
Copyright © 2014 Mancini and Sonis. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance with
accepted academic practice. No use, distribution or reproduction is permitted which
does not comply with these terms.
March 2014 | Volume 5 | Article 51 | 9